Intestine colonizing strains of lactobacilli

ABSTRACT

A process for isolation of a strain of Lactobacillus having the ability of being established on human intestinal mucosa in vivo and being able to remain therein after oral administration for at least 10 days after the completion of the administration. 
     By the process the new strains L. plantarum 299 (DSM 6595) and L. casei ssp. rhamnosus 271 (DSM 6594) have been isolated, which are useful for the prophylaxis or treatment of bacterial infections, especially in the form of a fermented nutrient composition.

This application is a 371 of PCT/SE92/00528, filed Jul. 24, 1992.

The present invention refers to a process for isolation of strains ofLactobacillus having the ability to colonize and become established onintestinal mucosa in vivo after oral administration, strains obtained bythis process, and the use thereof for the prophylaxis or treatment ofbacterial infections, especially in the form of a composition comprisingan oatmeal based nutrient solution fermented by one of said strains.

Many people have a disturbed intestinal microflora, that is, the balancebetween useful and harmful intestinal bacteria is disturbed. A number offactors, among others stress, the occurence of bile salts, diet, etc.influence the bacterial flora. Most important is, however, that modernantibiotic treatment can destroy the normal flora for a long period oftime, and thus, eliminate a normal fermentation process. Should thefermentation process be disturbed and the number of useful bacteria bereduced, the consequence will be that the colon mucosa withers away andceases to function at the same time as the potentially malignantbacteria rapidly grow in number. These bacteria penetrate themalfunctioning colon wall and infect the organs of the body which leadsto the so called intensive-care-disease with pus foci all over the bodyand possibly also an abolished function of most of the organs of thebody, a collapse of organs. Blood poisoning, sepsis, caused by abscessesin the abdominal cavity is still a very common surgical complication inconnection with abdominal surgery with a high death-rate. These patientsare today treated by administration of antibiotics and surgicaltreatment of the abscess to the extent it could be located. At presentantibiotics are conventionally administered before intestinal surgery inorder to reduce the risk of post-operative infections and illness causedthereby. However, the treatment with antibiotics is expensive andmoreover associated with a risk of different complications such asallergy and destruction of the normal intestinal flora and overgrowthwith still more pathogenic bacteria.

The fact that lactobacilli should have a favourable effect on theintestinal mucosa is an old idea which has been brought up again. Thereare however many unclear points as to which microorganisms are involvedand as to the ecology of the intestines. Another problem in thisconnection is that the classification of the genus Lactobacillus isincomplete which makes it difficult to identify those strains which arefavourable to the function of the intestines. What, after all, seems tobe commonly accepted today is that:

Bacteria of the genus Lactobacillus have a manifest ability ofpreventing the establishing of pathogenic bacteria in various ways,irrespective of foodstuffs or intestines being concerned;

Certain strains of Lactobacillus are more effective than other strainsof the same species in protecting and activating the intestines;

Foodstuffs fermented by lactobacilli have proven to have a cholesterolreducing effect, probably because of a checking of the cholesterolproduction in the intestines, but maybe also because the bacteria usecholesterol for the production of steroids;

The consumption of great amounts of lactobacilli improves the intestinalmotoric activity, the cause of this effect is unknown;

A large proportion of lactobacilli in the intestines counteracts cancer,something which seems to have several grounds. Firstly, certainlactobacilli are able to prevent the production of nitroseamines in theintestines by means of the enzyme nitritereductase; nitroseamines arecancerogenic. Secondly, lactobacilli may obstruct certain bacteriallyproduced enzymes in the intestines from activating potentiallycarcinogenic substances. Finally, there are indications of lactobacillihaving growth restricting effect on cancer rumours, maybe because themacrophages of the immunological defence system are activated by thepresence of the lactobacilli.

A decisive weakness of the lactobacilli used today in most conventionalfoodstuffs is the poor survival of these organisms during the passagethrough the stomach and duodenum. This brought about a the developmentof a product called "acidofilusfil", acidophilus sourmilk, wherein milkwas fermented with a strain of Lactobacillus acidophilus isolateddirectly from human faeces. L. acidophilus manages the passage throughthe upper part of the gastro-intestinal tract well. However, in order tohave an effect on the microflora in the intestines for a longer periodof time, it is essential that the lactobacillus is able to becomeestablished in the intestines. According to Lidbeck, A. et al, Scand JInfect Dis, 4, pp 531-537, 1987, the increase in the number oflactobacilli in the microflora of the intestines, which occurs afterconsumption of a preparation containing Lactobacillus acidophilus, isgradually slowing down as the consumption thereof ceases, andconsequently after 9 days without supply the bacterial flora hasregained its original composition.

EP-A2-0 199 535 describes a biologically pure culture of Lactobacillusacidophilus, ATCC accession No. 53 103, isolated from human faeces,being able to adhere to mucosal cells in tests in vitro. An adherence invivo has, however, not been demonstrated.

WO 89/05849 describes lactic acid bacteria isolated from thegastro-intestinal tract in pigs and selected by means of, among others,adhesion to gastro-intestinal epithelial cells from pigs in vitro andtolerance against acid and bile. Said bacteria can be used for thefermentation of milk which then can be given to piglets to prevent ortreat i.a. E. coli diarrhoea.

The strains of Lactobacillus which are commercially used today haveabove all been selected for being passably capable of growing in currentprimary products as for example milk. If a certain strain is to exercisean optional favourable influence, it is without doubt a prerequisitethat it is able to become established in the intestines and to competewith the existing microflora. Knowledge about which properties arenecessary for a certain Lactobacillus strain to be able to stand thiscompetition is for the most part unknown.

The present invention refers to a process for isolaton of a strain ofLactobacillus having the ability to colonize and become established onhuman intestinal mucosa in vivo, characterized in that lactobacilli areisolated from human intestinal mucosa and are pure cultured in asuitable nutritient medium and then selected as to the ability tocolonize and become established in the intestines.

The ability of the strain to colonize in the intestines is preferablytested by oral administration, and a subsequent verification of theoccurence on the intestinal mucosa at least 10 days after the completionof the administration.

A complementary selection of isolated strains can take place, before orafter the test of the colonization, by an evaluation of differentfunctional and technical properties, such as bile resistance,pH-resistance, ability of fermentation of a requested substrate,preferably oatmeal, and of producing flavour, ability to resistfreeze-drying, antibiotics resistance, etc.

To manage the passage through the gastro-intestinal tract the selectedstrains thus ought to be able to survive at a pH of 1.0 for 30 minutesand also to grow in the presence of 0.1% bile.

The invention also refers to strains of Lactobacillus having the abilityof colonizing human intestinal mucosa in vivo, obtained by the isolationprocess described above. According to one theory the strains ofLactobacillus which are facultatively heterofermentative constitute apreferred type for the establishment in the intestines.

The invention especially refers to new Lactobacillus strains having theability of colonizing human intestinal mucosa in vivo, which have beendeposited according to the Budapest Agreement at the DSM--DeutscheSammlung yon Mikroorganismen und Zellkulturen GmbH--, Braunschweig,Germany on Jul. 2, 1991, that is

    ______________________________________                                        Lactobacillus plantarum 299                                                                            DSM 6595                                             Lactobacillus casei ssp. rhamnosus 271                                                                 DSM 6594                                             ______________________________________                                    

The invention also refers to variants thereof having an essentiallycorresponding REA-pattern. A REA-pattern refers to the pattern formed inelectrophoresis on agar gel of DNA which has been decomposed with arestriction enzyme according to the method described below. Bycharacterization of the strains by means of their REA-pattern theidentity of the used isolates can be established, something which hasnot been possible before. Closely related strains of Lactobacillus withdifferences in the REA-pattern show differences as to the ability ofadherence to intestinal epithelium.

The invention also refers to a composition for the prophylaxis ortreatment of infections in the gastro-intestinal tract, which comprisesa Lactobacillus strain having the ability to colonize and becomeestablished in human intestinal mucosa in vivo, which has been obtainedaccording to the method of the invention, combined with a conventionalcarrier.

In particular the invention refers to a composition comprising any ofthe strains

    ______________________________________                                        Lactobacillus plantarum 299                                                                            DSM 6595                                             Lactobacillus casei ssp. rhamnosus 271                                                                 DSM 6594                                             ______________________________________                                    

or a variant thereof having an essentially corresponding REA-pattern.

Conventional carriers are for example physiologically acceptablesubstrates fermented by the bacterium in question, as well as foodstuffsof various kinds, especially based on starch or milk, but also inertsolid or liquid substances, such as saline or water. A suitablesubstrate should contain liquid or solid fibres which are not resorbedin the gastro-intestinal tract and which when fermented withLactobacillus form short fatty acids. As an example of suitable,starch-containing substrates can be mentioned cereals, such as oats andwheat, corn, root vegetables such as potatoes and certain fruits such asgreen bananas.

Modern medical care of patients in connection with illness and surgeryis to a large extent based on the supply of nutrition via the veins,whereby the intestines are not supplied with material to ferment withsubsequent consequences. The colon functions as the body's ownfermentation tank, the purpose of which is to produce useful nutrients,among others for the function of the colon itself, but also foreliminating harmful substances, for example heavy metals, excessiveamounts of cholesterol etc. In order for the colon to function theremust be a supply of suitable bacteria and substrates, particularlystarch and dietary fibres. About half of the contents of the colon isbacteria, mostly of the anaerobic type. The most important bacteria arethose located on the colon mucosa. Among the bacteria of the colon thereis a minority of a potentially harmful type. As long as the usefulbacteria are present the harmful bacterial flora is suppressed. Recentstudies have shown that the colon mucosa obtains most of its nutritionfrom fermentation products, mainly in the form of short fatty acids. Anormal fermentation process requires a supply of about 30 g of dietaryfibre daily and the presence of suitable bacteria.

A preferred substrate for the composition according to the invention,which also gives the composition an excellent nutritional value, is anutrient solution based on oatmeal. The cereal oats has shown to be agood sustrate for fermentation in many ways: It is rich in proteins,carbohydrates, fat, dietary fibre and also water-soluble fibre, socalled β-glucans. In addition oats or oatmeal fat has a very highcontent of surface-active phospholipids, which function as gastricmucosal barrier "corrosion inhibitors" and hence give mucosalprotection. Finally, the amino acid composition of oat proteinscorresponds to a large extent to the needs of the human body. In WO89/08405 a nutrient composition is described suitable for enteralfeeding, which is obtained by a combination of enzymatic decompositionof oatmeal with α-amylase, possibly protease, and β-glucanase and heattreatment and fermentation with a lactobacillus having the ability toadhere to the intestines spontaneously. The nutrient compositiondescribed in the referred patent application is in combination with aLactobacillus strain according to the invention an excellent compositionfor nutrient administration to patients in connection with the normaltreatment after large operations, for special treatment of patientsbeing victims of the intensive-care-disease or an organ collapse, andfor treatment of different intestinal diseases, for example ulcerativecolitis.

To be useful in an oatmeal based nutrient composition according to theinvention a Lactobacillus strain should fulfil the following conditions:

good fermentation of oats;

survival at a pH of 1.0 (which corresponds to the pH in the stomach) for30 minutes;

survival and growth in the presence of bile salts;

ability of settling and remaining on the intestinal mucosa.

It is also essential that the pH-value during the fermentation isreduced quickly in order to stop the growth of other bacteria.

It has been shown that the administration of lactobacilli having theability to colonize in or adhere to the intestines can suppress adifferent bacterial flora from colonizing the intestines and thus reducethe risk of sepsis in connection with bacterial infections such ascomplications following abdominal surgery. This treatment seems to be asefficient as the conventional treatment used today in the form ofantibiotics. Hence, it seems to be reasonable that patients who aresubjected to intestinal surgery are pretreated with lactobacilli ratherthan antibiotics. This means that a cheaper form of therapy could beestablished with less potential secondary effects as the normalintestinal flora would not be destroyed.

The invention also refers to the use of a nutrient composition fermentedby a Lactobacillus strain in accordance with the invention instead ofantibiotics for the prophylaxis or treatment of bacterial infections inconnection with surgical operations, post surgical rehabilitation etc.

It especially refers to an oatmeal based nutrient solution fermented byLactobacillus plantarum 299.

Experiments with test animals have shown a statistically valid survivalin animals treated with a composition comprising a Lactobacillus strainisolated from the intestinal mucosa of an animal of the same species.Tests with rats have shown a good prevention and quicker healing ofexperimentally induced colitis (colit) and ulcers in the intestines.

The composition according to the invention can be administered in anysuitable way, preferably orally or rectally, for example in the form ofenema. It can also be administered enterally through a catheter insertedin the intestines via the stomach or directly in the intestines. Testshave shown that the effect is improved if dietary fibres in the form offor example oatmeal gruel or of β-glucans are supplied. The treatmentshould take place once or several times daily for a period of 1-2 weeks.

BRIEF DESCRIPTION OF THE DRAWINGS

On the enclosed drawings FIGS. 1-2 show the REA-pattern of the newLactobacillus strains 299 and 271; and

FIG. 3 shows the concentration of lactobacilli in ileum before,immediately after, and a few days after, respectively, oraladministration of an oatmeal gruel fermented by Lactobacillus.

EXAMPLES

Isolation of Lactobacillus strains from humans

In order to isolate strains having the ability to colonize and becomeestablished on human intestinal mucosa, strains of Lactobacillus havebeen sampled from human mucosa. Biopsies from colon were taken by meansof enteroscopy and pieces of the intestinal mucosa from the smallintestine (jejunum and ileum) were removed in connection with surgicaloperations. The mucosa samples were immediately placed in a specialmedium (0.9% NaCl, 0.1% pepton, 0.1% Tween 80 and 0.02% cystine; allvalues refer to % by weight/volume), homogenized in ultrasonic baths for2 minutes and stirred for 1 minute before being placed on Rogosa agar(Difco Laboratories, Detroit, Mich., U.S.A.). The plates were incubatedanaerobically at 37° C. for 2 d (Gas Pak Anaerobic System, BBL). One tothree colonies were picked at random from each plate and were grown inpure cultures 5 to 9 times on Rogosa agar and kept as dense cultures ina frozen buffer at -80° C. A total of 209 Lactobacillus strains wereisolated from about 61 different subjects. All isolates werecharacterized as to the ability of fermenting 49 different carbohydratesby means of API 50 CH, a commercial test kit from API, Montalieu Verceu,France. No significant difference in the composition of the lactobacilliflora between the small and the large intestines could be found.

Representative strains from the different groups were evaluated as topH-resistance, ability of growing in the presence of bile, and abilityof fermenting oatmeal gruel.

The pH-resistance was tested by adding 0.1 ml bacterial suspension (10⁹CFU/ml which had been cultivated in Rogosa broth, centrifuged andresuspended in a physiological salt solution) to 2 ml phosphate bufferat pH 1.0. After 30 minutes Rogosa agar plates were inoculated and ifany growth was visible after incubation at 37° C. for 3 days the testwas considered to be positive. Only a few of the tested strains passedthis test.

Growth in the presence of bile was tested by growing isolates ofLactobacillus in the presence of 0.1% and 0.15%, respectively, beef bilein Rogosa agar plates incubated anaerobically for 3 days at 37° C. About80% of the strains were able to grow in the presence of 0.1% bile,whereas only 18% managed to grow in 0.15% bile.

Based on the results of these tests 20 different Lactobacillus strainswere selected for further investigation.

Intestinal colonization in vivo in humans

Healthy test subjects were for a certain period of time daily given afermented oatmeal gruel comprising a mixture of twenty different strainsof Lactobacillus, carefully selected in accordance with the above. Itwas then investigated which of the consumed strains could be found onthe mucosa from the small and large intestine.

Fermented oatmeal gruel was made according to the protocol describedbelow. This was done with each of the strains of Lactobacillus in thestudy, as stated in Table 1 below. The different preparations were mixedin such proportions that the final product contained 8×10⁷ CFU per gramfreeze-dried product.

In the study 12 volunteers aged between 31 and 56 years participated,each of which received ten bottles of 100 ml liquid oatmeal gruel basedon 1 g freeze-dried product per ml water. Samples from the intestinalmucosa were taken before the consumption of the oatmeal gruel hadstarted, after 11 days when the subjects had consumed 100 ml oatmealgruel for breakfast daily for a period of 10 days, and after another 10days, that is 11 days after the completion of the oatmeal gruelconsumption. The intestinal samples were taken as biopsies from thesmall intestine (ileum) by means of a Watson capsule, and from rectumwith a rectoscope. The biopsies were prepared as described above andanalysed as to the contents of viable Lactobacillus. From each sampleabout ten colonies were picked from the Rogosa agar plate, which weregrown in pure cultures and freeze-stored at -80° C. until they wereidentified.

All isolates were tested on API 50 CH as above. The isolates that seemedto correspond with or mainly correspond with any of the test strainswere tested further by plasmid analysis and restriction endonucleaseanalysis according to the methods described below.

As a general trend it was observed that the content of lactobacilli onthe intestinal mucosa was increased during the consumption of fermentedoatmeal gruel and that this increase was continued for 11 days after thecompletion of the administration. In FIG. 3 the logarithmicconcentration of lactobacilli in ileum is shown by means of a columndiagram before the start of the test (t=0), on the day after thecompletion of the test (t=1) and after another 10 days (t=11). Theincrease was more pronounced in the small intestine, but on the otherhand the content of lactobacilli as a whole was larger in the largeintestine. Furthermore, it could be noted that the contents of Gramnegative anaerobic bacteria in the colon were reduced after theconsumption of the fermented oatmeal gruel.

The following strains were found in a dominating position on theintestinal mucosa 10 days after the completion of the administration oflactobacilli:

Lactobacillus plantarum 299 was found in 11 subjects (in 5 subjects onlyon the small intestine and in 5 others only on the large intestine);

Lactobacillus casei ssp. rhamnosus 271 was found in 4 subjects (in 1only on the small intestine and in 2 others only on the largeintestine);

Lactobacillus reuteri 108 was found in 4 subjects (in 1 only on thesmall intestine and in 1 other only on the large intestine);

Lactobacillus murinus/casei ssp. tolerance 294 was found in 2 subjects.

The strains which were reisolated 11 days after the completedadministration were found on the mucosa in an approximate concentrationof 3×10³ to 10⁵ CFU/g mucosa for the small intestine and a concentrationof 10³ to 3×10⁷ CFU/g mucosa for the large intestine.

Preparation of oatmeal gruel

Fermented oatmeal gruel was made in three steps:

(i) 1295 g oatmeal (MP-450, Nord-Mills, Jarna; protein content 14.2% andash content 2.1%), 129.5 g enzyme mixture (Nord Malt, Soderhamn) and5390 g tap water were mixed and heated to 95° C. during slow stirring.The gruel was cooled to 50° C., 1% β-glucanase (weight/volume) was added(GV-L; Grindsted Products A/S, Braband, Denmark) and then was incubatedfor 2 hours at 50° C.;

(ii) The gruel was inoculated with fresh lactobacilli and fermented at37° C. for 15-20 hours. The pH was 3.4 to 3.9. The fermentation wascarried out with the different strains each separately and the number ofcolony forming units, CFU, per ml product varied between 6×10⁶ and 2×10⁸on Rogosa agar (anaerobically at 37° C. for 20 hours);

(iii) The fermented gruel was freeze-dried. The different products weremixed in such a proportion that the same value of CFU/g was obtained forall the strains. The mixture was supplemented with 20% (w/w) soybeanflour (protein 51%, ash content 5.5%, fat 1%). The enriched mixturecontained 2×10⁷ CFU/g and was kept at -18° C. Non-fermented

oatmeal gruel was made in the same way as above, but withoutfermentation.

Oatmeal gruel was made with all the 20 strains which had been selectedfor the intestine colonization test described above and was evaluated asto the concentration before and after freeze-drying and as to flavour.The results are given in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Selected strains of Lactobacillus for clinical tests                          Strain                                                                        No.   Description  CFU/g     CFU*/g  Flavour***                               ______________________________________                                        138   "aggregating"                                                                              8,8 × 10.sup.8                                                                    1,78 × 10.sup.8                                                                 1                                        132   L. salivarius                                                                              1,1 × 10.sup.8                                                                    6,5 × 10.sup.6                                                                  3                                         47   L. reuteri   1,2 × 10.sup.9                                                                    3,7 × 10.sup.7                                                                  1                                        108   L. reuteri   1,5 × 10.sup.9                                                                    1,88 × 10.sup.7                                                                 1                                         98   L. casei pseudo-                                                                           1,63 × 10.sup.9                                                                   6,6 × 10.sup.8                                                                  2                                              plantarum                                                               292   L. gasseri   1,58 × 10.sup.9                                                                   5,1 × 10.sup.8                                                                  4                                        299   L. plantarum 1,92 × 10.sup.9                                                                   6,71 × 10.sup.8                                                                 5                                        136   L. casei casei                                                                             3,5 × 10.sup.9                                                                    1,48 × 10.sup.9                                                                 2                                        Al    L. plantarum 2,27 × 10.sup.9                                                                   4,15 × 10.sup.8                                                                 5                                        271   L. casei     4,3 × 10.sup.9                                                                    6,10 × 10.sup.8                                                                 4                                              rhamnosus                                                               227   L. buchneri  9,45 ×  10                                                                        1,81 × 10.sup.8                                                                 1                                        140   L. gasseri   1,2 × 10.sup.8                                                                    8,5 × 10.sup.6                                                                  4                                        294   L. murinus/casei                                                                           1,63 × 10.sup.9                                                                   1,3 × 10.sup.8                                                                  3                                              tolerance                                                               283   L. plantarum 7,43 × 10.sup.8                                                                   7,55 × 10.sup.7                                                                 4                                        282   cluster 25** 7,8 × 10.sup.8                                                                    6,65 × 10.sup.7                                                                 2                                         96   cluster 19** 4,9 × 10.sup.8                                                                    4,3 × 10.sup.7                                                                  3                                         99   cluster 12** 4,6 × 10.sup.9                                                                    1,39 × 10.sup.9                                                                 4                                         99*  cluster 12** 1,0 × 10.sup.9                                                                    1,6 × 10.sup.8                                                                  2                                        308   L. acidophilus                                                                             5,9 × 10.sup.8                                                                    1,0 × 10.sup.8                                                                  3                                        280   L. salivarius                                                                              3,0 × 10.sup.8                                                                    2,43 × 10.sup.6                                                                 3                                        ______________________________________                                         *after freezedrying                                                           **the clusternumbering refers to a work in numerical taxonomy on intestin     associated lactobacilli by Molin G et al (under publication).                 ***on a scale 51                                                         

The ability of giving the oatmeal gruel a pleasant flavour by thefermentation was judged by an "expert panel" consisting of four personswho judged the oatmeal gruels fermented by different strains. Theflavour was estimated in a dropping scale from 5 to 1, where 5 denotesthe judgement "very good" and 1 the judgement "unsavoury". The valuesfor the 20 selected test strains are shown in Table 1 above.

Fermentation of oatmeal gruel

The four strains which were found on the intestinal mucosa in adominating amount were investigated further as to the ability to fermentoatmeal gruel, the ability to resist freeze-drying and as to thedevelopment of flavour in oatmeal gruel.

The ability of fermenting oatmeal gruel was judged by means of theability to reduce pH below 4.0 and form CFU at a level of >10⁸ CFU/g wetweight.

The ability of resisting freeze-drying in oatmeal gruel was anotherselection criterium. In this connection the CFU concentration wasmeasured after freeze-drying.

The result of the test above with oatmeal gruel is shown in table 2below.

                  TABLE 2                                                         ______________________________________                                        Fermentation of oatmeal gruel with selected strains of                        Lactobacillus                                                                           Strain 299                                                                            271      294      108                                       ______________________________________                                        final pH    3.6       3.8      3.4    3.8                                     acid value  8.0       6.5      8.1    6.5                                     L-lactate, g/100 g                                                                        0.18      0.40     0.32   0.25                                    D-lactate, g/100 g                                                                        0.390     0.031    0.24   0.19                                    lactate tot., g/100 g                                                                     0.57      0.43     0.55   0.44                                    D-lactate in %                                                                            69        7        43     44                                      acetate, g/100 g                                                                          0.0084    0.013    0.13   0.0026                                  reduction after                                                                           65        86       94     98                                      freeze-drying in %                                                            final CFU/g 2 × 10.sup.9                                                                      4 × 10.sup.9                                                                     8 × 10.sup.8                                                                   1 × 10.sup.9                      ______________________________________                                    

In addition the flavour of the selected 4 strains was evaluated incomparision with on one hand a commercial youghurt culture(Streptococcus thermophilus and Lactobacillus bulgaricus) and on theother hand a commercial culture of acidophilus sourmilk (Lactobacillusacidophilus) using the same evaluation symbols as above. The results aregiven in Table 3 below.

                  TABLE 3                                                         ______________________________________                                        Flavour of oatmeal gruel fermented with strains of                            Lactobacillus                                                                          Acidophilus                                                          Yoghourt sourmilk    299    271    294  108                                   ______________________________________                                        3        2           5      4      3    1                                     ______________________________________                                    

On the basis of these values the strains 299 and 271 were judged to beof special interest and are described in further detail below.

Description of Lactobacillus strains 299 and 271

The strains 299 and 271, which were both isolated from healthy humanintestinal mucosa, have been deposited at the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH on Jul. 2, 1991 and have beengiven the deposition numbers DSM 6595 (299) and DSM 6594 (271).

Phenotype description

The strains 299 and 271 are Gram positive, catalase negative rodsgrowing on Rogosa agar at pH 5.5. The capacity of the strains to fermentdifferent carbohydrates is shown in Table 4. The tests have been carriedout by means of the API 50 CH in accordance with the instructions of themanufacturer.

                  TABLE 4                                                         ______________________________________                                        Ability to form acid from different carbohydrates                                                  Strains                                                                       299  271                                                 ______________________________________                                         1.       Glycerol         -      -                                            2.       Erythrithol      -      -                                            3.       D-arabinose      -      -                                            4.       L-arabinose      +      -                                            5.       Ribose           +      +                                            6.       D-xylose         -      -                                            7.       L-xylose         -      -                                            8.       Adonithol        -      -                                            9.       β-methyl-xyloside                                                                         -      -                                           10.       Galactose        +      +                                           11.       D-glucose        +      +                                           12.       D-fructose       +      +                                           13.       D-mannose        +      +                                           14.       L-sorbose        -      +                                           15.       Rhamnos          -      +                                           16.       Dulcitol         -      -                                           17.       Inositol         -      +                                           18.       Mannitol         +      +                                           19.       Sorbitol         +      +                                           20.       α-methyl-D-mannoside                                                                     +      +                                           21.       α-methyl-D-glucoside                                                                     -      +                                           22.       N-acetyl-glucosamine                                                                           +      +                                           23.       Amygdalin        +      +                                           24.       Arbutin          +      +                                           25.       Esculin          +      +                                           26.       Salicin          +      +                                           27.       Cellobiose       +      +                                           28.       Maltose          +      +                                           29.       Lactose          +      +                                           30.       Melibiose        +      -                                           31.       Saccharose       +      +                                           32.       Trehalose        +      +                                           33.       Inulin           -      -                                           34.       Melezitose       +      +                                           35.       D-raffinose      -      -                                           36.       Amidon           -      -                                           37.       Glycogene        -      -                                           38.       Zylitol          -      -                                           39.       β-gentiobiose                                                                             +      +                                           40.       D-turanose       +      +                                           41.       D-lyxose         -      +                                           42.       D-tagatose       -      +                                           43.       D-fucose         -      -                                           44.       L-fucose         -      -                                           45.       D-arabitol       -      -                                           46.       L-arabitol       -      -                                           47.       Gluconate        +      +                                           48.       2-keto-gluconate -      -                                           49.       5-keto-gluconate -      -                                           ______________________________________                                    

Phenotypically strain 299 can be identified as Lactobacillus plantarum(only raffinose deviated from the test pattern for L. plantarum ATCC14917T; this is the type strain for the species L. plantarum, that isthe strain which defines the species). 271 can be identified asLactobacillus casei subsp. rhamnosus (corresponds completely to the typestrain for the species).

Genotype description

The two strains have been examined as to the cleavage pattern of thechromosome DNA in connection with cleavage with EcoRI, throughrestriction-endonuclease analysis--REA--(method according to Ståhl M.,Molin G., Persson A., Ahrne S. & Ståhl S., International Journal ofSystematic Bacteriology, 40:189-193, 1990). Schematically REA can bedescribed as follows:

(1) Chromosome DNA is isolated from the strains involved in the study;

(2) The DNA is cleaved with restriction enzymes;

(3) The cleaved DNA fragments are separated as to size by agarose gelelectrophoresis;

(4) The band patterns of the different strains are registered andinterpreted by means of a laser densitometer and associated programs.The differences between the strains regarding the REA-pattern can beexpressed mathematically by means of principal component analysis.1990).

Furthermore an examination has been carried out referring to thecontents of plasmids (method according to Ahrne S., Molin G. & Ståhl S.,Systematic and Applied Microbiology 11:320-325, 1989).

Strain 299: This strain contains four plasmids which are of the sizes of4 MDal, 9 MDal, 20 MDal and 35 MDal, respectively. The cleavage patternof the chromosomal DNA is shown in FIG. 1. The lane marked with 299shows the pattern of strain 299 and the lanes marked with a v representa genetical variant of strain 299 from two different isolates; thisvariant was one of the 20 strains that were tested on humans and has inTable 1 been denoted as A1; lane s denotes the standard, High M_(w) DNAMarkers (AEH; BRL, Bethesda Research Laboratories, Life Technologies,Inc.). The variant of 299 can by means of common phenotype tests not beseparated from 299. Also genetically 299 and 299v are very close. Thevariant has also proved to have the same ability to be established inhuman intestinal mucosa.

Strain 271: This strain contains two plasmids with a size of 3 MDal and5 MDal, respectively. The cleavage pattern of the chromosomal DNA of thestrain is shown in FIG. 2, as lane A; lane v shows a genetical variantof strain 271; and lane s denotes the same standard as in FIG. 1. Thevariant of 271 can with common phenotype tests not be separated from271. Also genetically 271 and 271v are very close. The variant also hasturned out to have the same ability to colonize the human intestinalmucosa as the sister strain.

Genetically the two examined strains differ essentially. They alsodiffer significantly from the respective type strain.

Cultivation of Lactobacillus 299

An inoculate from a freezer of -80° C. is added to 50 ml LactobacillusCarrying Medium (LCM, Efthymiou & Hansen, J. Infect. Dis., 110:258-267,1962) or Rogosa,

is incubated for about 40 hours at 37° C.,

50 ml is inoculated into 500 ml LCM,

is incubated about 40 hours at 37° C.,

500 ml is inoculated into 5 liters,

is incubated about 25-30 hours at 37° C.,

is centrifuged at 10 000 rpm for 10 minutes,

is washed once in a physiological salt solution,

the pellet is dissolved in about 1 liter of physiological salt solution.

This amount is estimated to be sufficient for about 400-500 l of oatmealgruel. Cultivation media are not optimized. Rogosa worked better thanLCM, possibly due to a better buffer function. 2% glucose was added toLCM. The same procedure can be used for producing the otherLactobacillus strains.

Biological test on rat

Rats having a weight of 250-300 g were subjected to a standard operationto develop an abscess in the abdominal cavity by isolating andpuncturing a part of the large intestine by which a constant leekage ofintestinal contents out into the abdominal cavity was obtained whichcaused an abscess within 24 hours, sepsis and subsequent high rate ofmortality. Three groups of 30 animals each were used. Group 1 was anuntreated control group, Group 2 was treated with antibiotics, byinjection, and Group 3 was supplied with lactobacilli in the form of afermented oatmeal gruel to the stomach. The Lactobacillus strain whichwas used had been isolated from rat intestinal mucosa and in testsproved to be able to colonize and become established in rat intestines.

Evaluation of the test was made by analysis of the content of bacteriain the blood, something which is equivalent to sepsis, as well ascultures from the abdominal cavity and intestines. The result shows thatall animals in Group 1 had bacteria in the blood, which should lead to avery high rate of mortality. In Groups 2 and 3 similar results wereobtained with the occurrence of bacteria in 3 of 30 animals, however, toa much lesser extent than in Group 1.

We claim:
 1. A biologically pure culture of a Lactobacillus strain having the ability to colonize human intestinal mucosa in vivo and having all the identifying characteristics of Lactobacillus plantarum 299 DSM 6595 or Lactobacillus ssp rhamnosus 271 DSM
 6594. 2. The biologically pure culture of claim 1, wherein said strain is Lactobacillus plantarum 299 DSM
 6595. 3. The biologically pure culture of claim 1, wherein said strain is Lactobacillus ssp rhamnosus 271 DSM
 6594. 